Gothot A, van der Loo J C, Clapp D W, Srour E F
Division of Hematology/Oncology, Department of Medicine, and Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana School of Medicine, Indianapolis, IN, USA.
Blood. 1998 Oct 15;92(8):2641-9.
Most primitive hematopoietic progenitor cells reside in vivo within the G0/G1 phase of the cell cycle. By simultaneous DNA/RNA staining it is possible to distinguish G0 and G1 states and to isolate cells in defined phases of the cell cycle. We report here the use of cell cycle fractionation to separate human mobilized peripheral blood (MPB) CD34(+) cells capable of repopulating the bone marrow (BM) of non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice. In freshly isolated MPB, repopulating cells were predominant within the G0 phase, because transplantation of CD34(+) cells residing in G0 (G0CD34(+)) resulted on average in a 16.6- +/- 3.2-fold higher BM chimerism than infusion of equal numbers of CD34(+) cells isolated in G1. We then investigated the effect of ex vivo cell cycle progression, in the absence of cell division, on engraftment capacity. Freshly isolated G0CD34(+) cells were activated by interleukin-3 (IL-3), stem cell factor (SCF), and flt3-ligand (FL) for a 36-hour incubation period during which a fraction of cells progressed from G0 into G1 but did not complete a cell cycle. The repopulating capacity of stimulated cells was markedly diminished compared with that of unmanipulated G0CD34(+) cells. Cells that remained in G0 during the 36-hour incubation period and those that traversed into G1 were sorted and assayed separately in NOD/SCID recipients. The repopulating ability of cells remaining in G0 was insignificantly reduced compared with that of unstimulated G0CD34(+) cells. On the contrary, CD34(+) cells traversing from G0 into G1 were largely depleted of repopulating capacity. Similar results were obtained when G0CD34(+) cells were activated by the combination of thrombopoietin-SCF-FL. These studies provide direct evidence of the quiescent nature of cells capable of repopulating the BM of NOD/SCID mice. Furthermore, these data also demonstrate that G0-G1 progression in vitro is associated with a decrease in engraftment capacity.
大多数原始造血祖细胞在体内处于细胞周期的G0/G1期。通过同时进行DNA/RNA染色，可以区分G0和G1状态，并分离处于细胞周期特定阶段的细胞。我们在此报告使用细胞周期分级分离法来分离能够重建造血功能的人动员外周血（MPB）CD34(+)细胞，这些细胞可重建造血功能的人动员外周血（MPB）CD34(+)细胞，这些细胞可重建非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠的骨髓（BM）。在新鲜分离的MPB中，能够重建造血功能的细胞在G0期占主导地位，因为移植处于G0期的CD34(+)细胞（G0CD34(+)）平均导致的骨髓嵌合率比输注等量处于G1期分离的CD34(+)细胞高16.6±3.2倍。然后，我们研究了在无细胞分裂情况下体外细胞周期进程对植入能力的影响。新鲜分离的G0CD34(+)细胞用白细胞介素-3（IL-3）、干细胞因子（SCF）和fms样酪氨酸激酶3配体（FL）激活，孵育36小时，在此期间一部分细胞从G0期进入G1期，但未完成一个细胞周期。与未处理的G0CD34(+)细胞相比，刺激后细胞的重建造血功能明显降低。在36小时孵育期内仍处于G0期的细胞和进入G1期的细胞被分选并分别在NOD/SCID受体中进行检测。与未刺激的G0CD34(+)细胞相比，仍处于G0期的细胞的重建造血能力略有降低。相反，从G0期进入G1期的CD34(+)细胞的重建造血能力大大降低。当G0CD34(+)细胞用血小板生成素-SCF-FL联合激活时，也得到了类似的结果。这些研究提供了直接证据，证明能够重建造血功能的细胞具有静止特性。此外，这些数据还表明，体外G0-G1期进展与植入能力下降有关。
